Biochem. J. (2009) 421, 29–42 (Printed in Great Britain) doi:10.1042/BJ20090489 29

Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

Juan M. GARC´IA-MART´INEZ*, Jennifer MORAN†, Rosemary G. CLARKE‡, Alex GRAY§, Sabina C. COSULICH, Christine M. CHRESTA and Dario R. ALESSI*1 *MRC Protein Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., †Division of Therapy, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., ‡Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., §Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K., and Cancer Bioscience, Astrazeneca, Alderley Park, Cheshire, SK10 4TG, U.K.

mTOR (mammalian target of rapamycin) stimulates cell growth of Thr308 and/or induces a conformational change that protects by phosphorylating and promoting activation of AGC (protein Thr308 from dephosphorylation. In contrast, Ku-0063794 does A/ G/) family such not affect Thr308 phosphorylation in fibroblasts lacking essential as Akt (), S6K (p70 ) and SGK mTORC2 subunits, suggesting that signalling processes have (serum and glucocorticoid protein kinase). mTORC1 (mTOR adapted to enable Thr308 phosphorylation to occur in the absence complex-1) phosphorylates the hydrophobic motif of S6K, of Ser473 phosphorylation. We found that Ku-0063794 induced whereas mTORC2 phosphorylates the hydrophobic motif of Akt a much greater dephosphorylation of the mTORC1 substrate and SGK. In the present paper we describe the small molecule 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than Ku-0063794, which inhibits both mTORC1 and mTORC2 with rapamycin, even in mTORC2-deficient cells, suggesting a form an IC50 of ∼10 nM, but does not suppress the activity of 76 other of mTOR distinct from mTORC1, or mTORC2 phosphorylates protein kinases or seven lipid kinases, including Class 4E-BP1. Ku-0063794 also suppressed cell growth and induced

1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher aG1-cell-cycle arrest. Our results indicate that Ku-0063794 will concentrations. Ku-0063794 is cell permeant, suppresses be useful in delineating the physiological roles of mTOR and activation and hydrophobic motif phosphorylation of Akt, S6K may have utility in treatment of cancers in which this pathway is and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not inappropriately activated. regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3- Key words: Akt/protein kinase B (PKB), cancer, kinase inhibitor, phosphoinositide-dependent protein kinase-1). We interpret this phosphoinositide 3-kinase (PI3K), p70 ribosomal S6 kinase as implying phosphorylation of Ser473 promotes phosphorylation (S6K), serum and glucocorticoid protein kinase (SGK).

INTRODUCTION activated protein kinase) pathway [11]. mTORC2 consists of mTOR, Rictor, Sin1, mLST8 as well as Protor and is insensitive The mTOR (mammalian target of rapamycin) protein kinase to acute rapamycin treatment [12–18]. The activity of mTORC2 lies at the nexus of signalling pathways that regulate cell activity is controlled by PI3K, but is insensitive to amino acids or growth and proliferation. Many cancer-driving mutations in energy stress. such as receptor tyrosine kinases, Ras, PI3K (phosphoinositide A key role of mTOR complexes is to phosphorylate and 3-kinase) and PTEN ( and tensin homologue deleted control the activity of a subgroup of related AGC (protein kinase on 10) stimulate proliferation, growth and survival A/protein kinase G/protein kinase C) family kinases that comprise by activating mTOR kinase. Two mTOR complexes have isoforms of Akt, S6K (p70 ribosomal S6 kinase), PKC (protein been characterized, termed mTORC1 (mTOR complex-1) and kinase C) and SGK (serum and glucocorticoid protein kinase). mTORC2 (reviewed in [1,2]). mTORC1 consists of mTOR, mTOR complexes phosphorylate these at a conserved Raptor and mLST8 and is acutely inhibited by rapamycin [3–5]. C-terminal non-catalytic residue, termed the ‘hydrophobic motif’. mTORC1 is activated by insulin and growth factors via a PI3K- Activation of AGC kinases also requires phosphorylation of controlled pathway, involving Akt-mediated phosphorylation of an additional site within their kinase catalytic domain termed PRAS40 (proline-rich Akt substrate of 40 kDa) and tuberous the ‘T-loop motif’. This phosphorylation is executed by the sclerosis complex-2 protein, and activation of the Rheb GTPase PDK1 (3-phosphoinositide-dependent protein kinase-1) [19]. [6,7]. mTORC1 is also stimulated by amino acids through a mTORC1 phosphorylates the hydrophobic motif of S6K [4,5], pathway involving bidirectional transport of amino acids [8] and whereas mTORC2 phosphorylates the hydrophobic motifs of Akt the Rag GTPase [9,10]. Low-energy conditions suppress cell [20], SGK [21] and certain isoforms of PKC [17,22]. In the growth by inhibiting mTORC1 via the LKB1–AMPK (AMP- case of S6K, SGK and PKC isoforms, hydrophobic motif

Abbreviations used: AGC family, /protein kinase G/protein kinase C family; AMPK, AMP-activated protein kinase; DTT, dithiothreitol; eIF4E, eukaryotic initiation factor 4E; 4E-BP1, eIF4E-binding protein 1; ERK, extracellular-signal-regulated kinase; GST, glutathione ; HEK-293 cell, human embryonic kidney 293 cell; IGF, insulin-like growth factor; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; mTOR, mammalian target of rapamycin; mTORC, mTOR complex; NDRG1, N-Myc downstream-regulated -1; PDK, 3-phosphoinositide-dependent protein kinase; PH domain, pleckstrin homology domain; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PRAS40, proline-rich Akt substrate of 40 kDa; RSK, ribosomal S6 kinase; S6K, p70 ribosomal S6 kinase; SGK, serum and glucocorticoid protein kinase; SPHK, sphingosine kinase;. 1 To whom correspondence should be addressed (email [email protected]).

c The Authors Journal compilation c 2009 Biochemical Society 30 J. M. Garc´ıa-Mart´ınez and others phosphorylation creates a high-affinity docking site for PDK1, termed pNDRG1 3 × Thr-P) was raised against the nonapeptide thereby promoting phosphorylation and activation of these RSRSHpTSEG, whose sequence is common to all three SGK1 enzymes by PDK1 [19,23]. In the case Akt, interaction of phosphorylation sites on NDGR1 (used for immunoblotting). the PH (pleckstrin homology) domains in PDK1 and Akt with Anti-Akt1 (S695B, 3rd bleed; residues 466 – 480 of human Akt1

PtdIns(3,4,5)P3 (a product of PI3K activation), at the plasma RPHFPQFSYSASGTA, used for immunoblotting and immun- membrane of cells, brings these enzymes together and induces a oprecipitation), anti-S6K (S417B, 2nd bleed; residues 25–44 of conformational change in Akt that enables it to be phosphorylated human S6K, AGVFDIDLDQPEDAGSEDEL, used for immun- at Thr308 by PDK1 [24,25]. Recent data also indicates that oblotting and immunoprecipitation). Anti-GST (S902A, 1st bleed) mTORC2 controls the phosphorylation of Akt and PKC isoforms was raised against the GST tag expressed from pGex4T (used for at a conserved phosphorylation site termed the ‘turn motif’ immunoblotting). Anti-PRAS40 (S115B, 1st bleed; residues 238– that stabilizes these enzymes in an active conformation [26,27]. 256 of human PRAS40, DLPRPRLNTSDFQKLKRKY, used mTORC1 also phosphorylates other substrates such as the for immunoblotting). An antibody that recognizes PRAS40 eukaryotic translation initiation factor 4E-BP1 [eIF4E (eukaryotic phosphorylated at Thr246 (S114B, 2nd bleed) was raised against initiation factor 4E)-binding protein 1]. Phosphorylation of 4E- the residues 240–251 of human PRAS40, CRPRLNTpSDFQK BP1 by mTOR induces dissociation from eIF4E, promoting (used for immunoblotting). Anti-RSK2 (ribosomal S6 kinase 2) initiation of protein translation that is required to power cell (S382B, 1st bleed; residues 712–734 of human RNQSPVLEPV- growth and proliferation ([4,5],[28], but see [28a]). In the present GRSTLAQRRGIKK, used for immunoblotting and immuno- study, we describe the characterization of Ku-0063794, a novel precipitation); Anti-FoxO1 (S504A, 1st bleed) was made in sheep potent and highly specific small-molecule inhibitor of the mTOR using recombinant GST-fusion FoxO1 (2–655, used for im- protein kinase. We demonstrate that Ku-0063794 inhibits both munoblotting). Anti-β-tubulin (H-235, #sc-9104), phospho-RSK mTORC1 and mTORC2 in vitro and in vivo and can be employed Thr227 (#sc-12445-R) and the total mTOR antibody (#sc-1549) to dissect cellular functions of the mTOR pathway. were purchased from Santa Cruz Biotechnology. For phospho- immunoblotting of the hydrophobic motif of SGK1(Ser422), we 389 MATERIALS AND METHODS employed the Thr S6K antibody (# 9205) from Cell Signaling Technology, which we previously demonstrated cross-reacted Materials with the phosphorylated Ser422 of SGK1 [30]. The phospho- 473 308 450 Protein G–Sepharose and glutathione–Sepharose were purchased Akt Ser (#9271), Thr (#4056), Thr (#9267), phospho-S6K 389 235 from Amersham Bioscience. [γ -32P]ATP was from PerkinElmer; Thr (#9234), phospho S6 ribosomal protein Ser (#4856), IGF1 (insulin-like growth factor) was from Invitrogen. Tween total S6 ribosomal protein (#2217), 4E-BP1 total (#9452), 37 46 65 20, DMSO, PMA and dimethyl pimelimidate were from Sigma, phospho 4E-BP1 Thr /Thr (#9459), phospho 4E-BP1 Ser and CHAPS and rapamycin were from Calbiochem. Akti-1/2, (#9451), phospho-ERK (extracellular-signal-regulated kinase) 202 204 573 PI-103 and PD0325901-CL were synthesized by Dr Natalia Thr /Tyr (#9101), total ERK (#9102), phospho-RSK Thr 380 Shpiro at the MRC Protein Phosphorylation Unit, University of (#9346), phospho-RSK Thr (#9341), phospho-FoxO1/O3 24/32 α β 21 9 Dundee. Ku-0063794 was synthesized at AstraZeneca. The wild- Thr (#9464), phospho-GSK3 / Ser /Ser (#9331), the 2481 2448 type control mLST8+/+ and mLST8−/− knockout MEFs (mouse phospho-mTOR Ser (#2974) and the phospho-mTOR Ser embryonic fibroblasts) were described previously [17] and pro- (#2971) were purchased from Cell Signaling Technology. vided by David Sabatini (Whitehead Institute for Biomedical For phospho-immunoblotting of the phosphorylated T-loop of Research, Cambridge, MA, U.S.A.). The wild-type control S6K, we employed the pan-PDK1 site antibody from Cell Rictor+/+ and Rictor−/− knockout MEFs were described pre- Signaling Technology (#9379) as previously described [31].The α β viously [29] and provided by Mark Magnuson (Vanderbilt GSK3 / antibody (44–610) was purchased from Biosource. University School of Medicine, Nashville, TN, U.S.A.). The wild- The secondary antibodies coupled to horseradish peroxidase used type control Sin1+/+ and Sin1−/− knockout MEFs were described for immunoblotting were obtained from Thermo Scientific. previously [16] and provided by Bing Su (Yale University School General methods of Medicine, New Haven, CT, U.S.A.). Tissue culture, immunoblotting, restriction digests, DNA Antibodies ligations and other recombinant DNA procedures were performed using standard protocols. DNA constructs used for transfection The following antibodies were raised in sheep and affinity- were purified from E. coli DH5α using Qiagen plasmid Mega purified on the appropriate antigen: anti-mLST8 (S837B, 3rd or Maxi kits according to the manufacturer’s protocol. All bleed) was raised against the human full-length mLST8 protein DNA constructs were verified by DNA sequencing, which expressed in Escherichia coli (used for immunoblotting), anti- was performed by The Sequencing Service, School of Life Raptor (S682B, 3rd bleed; residues 1–20 of human Raptor, Sciences, University of Dundee, Dundee, U.K., using DYEnamic MESEMLQSPLLGLGEEDEAD, used for immunoblotting and ET terminator chemistry (Amersham Biosciences) on Applied immunoprecipitation), anti-Rictor (S654B, 3rd bleed; residues Biosystems automated DNA sequencers. For transfection studies, 6–20 of human Rictor, RGRSLKNLRVRGRND, used for typically ten 10-cm-diameter dishes of HEK-293 cells were immunoblotting in HEK-293 (human embryonic kidney 293) cultured, and each dish was transfected with 5–10 μgofthe cells and immunoprecipitation), anti-Rictor (S274C, 1st bleed; indicated plasmids using the polyethylenimine method [32]. residues 6–20 of mouse Rictor, RGRSLKNLRIRGRND, used for Cellular levels of PtdIns(3,4,5)P3 were measured as described immunoblotting), anti-Sin1 (S8C, 1st bleed) was raised against previously [33]. MEFs were cultured with additional non-essential the human full-length Sin1 protein expressed in E. coli (used for amino acids and 1% sodium pyruvate solution. immunoblotting). Anti-NDRG1 (N-Myc downstream-regulated gene-1) (S276B, 2nd bleed) was made in sheep using recombinant GST (glutathione transferase) fusion of full-length NDRG1 (used Buffers for immunoblotting). An antibody that recognizes NDRG1 The following buffers were used: Tris lysis buffer [50 mM phosphorylated at Thr346,Thr356 and Thr366 (S911B, 2nd bleed; Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 0.3% (w/v)

c The Authors Journal compilation c 2009 Biochemical Society Novel small molecule mTOR inhibitor 31

CHAPS, 1 mM sodium orthovanadate, 10 mM sodium β- was assayed against choline in a final volume of 50 μl containing glycerophosphate, 50 mM sodium fluoride, 5 mM sodium 25 mM glycine/NaOH, pH 8.5, 67 mM KCl, 5 mM MgCl2,1mM pyrophosphate, 0.27 M sucrose, 0.15 M NaCl, 0.1% (v/v) 2- choline, 1 μM ATP and 1 mM DTT and incubated for 30 min at mercaptoethanol, 1 mM benzamidine and 0.1 mM PMSF], Buffer room temperature. These three assays were stopped by addition A [50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA and 0.1% (v/v) of 50 μl Kinase Glo Plus Reagent, incubated for 10 min at room 2-mercaptoethanol], Hepes lysis buffer [40 mM Hepes, pH 7.5, temperature and read for 1 s/well. 120 mM NaCl, 1 mM EDTA, 0.3% (w/v) CHAPS, 10 mM Class 1 PI3Kα wasassayedasfollows:PI3Kα (diluted in sodium pyrophosphate, 10 mM sodium β-glycerophosphate, 50 mM Hepes, pH 7.5, 150 mM NaCl, 0.02% sodium cholate 50 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1 mM and 1 mM DTT) was assayed against PtdIns(4,5)P2 diC8 in a final benzamidine and 0.1 mM PMSF], Hepes kinase buf- volume of 50 μl containing 37 mM Hepes, pH 7.5, 111 mM NaCl, fer (25 mM Hepes, pH 7.5, 50 mM KCl and TBS-Tween Buffer 0.02% sodium cholate, 5 mM DTT, 5 mM MgCl2,1mMATP [50 mM Tris/HCl, pH 7.5, 0.15 M NaCl and 0.1% (v/v) Tween and 2 μM PtdIns(4,5)P2 and incubated for 70 min at room 20] and Sample Buffer [50 mM Tris/HCl, pH 6.8, 6.5% (v/v) temperature. Assays were stopped by addition of a 5.5 μl glycerol, 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol]. solution of 50 mM EDTA and 0.02% (w/v) sodium cholate. An aliquot (25 μl) of the resultant mixture was transferred Cell lysis to a Lumitrac 200 plate. Detection mix 1 [41 mM Hepes, pH 7.5, 123 mM NaCl, 1.7 μg GST–GRP1 (guanine-nucleotide- HEK-293, HeLa (human cervical carcinoma) or MEF cells were releasing protein 1), 0.16 μM PtdIns(3,4,5)P3–biotin, 1.6 μg cultured and treated as described in the Figure legends. Following streptavidin–allophycocyanin and 0.96 μg/ml europium-chelate- treatment, cells were rapidly rinsed twice with ice-cold PBS labelled antibody] was added to give a final volume of 50 μland and then lysed using Tris lysis buffer. Whole-cell lysates were ◦ incubated for 20 min at room temperature before reading. centrifuged (18000 g at 4 C for 20 min), supernatants were re- β β − ◦ Class 1 PI3K was assayed as follows: PI3K (diluted in moved and stored in aliquots at 80 C until required. 50 mM Hepes, pH 7.5, 150 mM NaCl, 0.02% sodium cholate and 1 mM DTT) was assayed against PtdIns(4,5)P2–diC8 in a final Specificity kinase panel volume of 50 μl containing 37 mM Hepes, pH 7.5, 111 mM NaCl, All assays were performed at The National Centre for 0.02% sodium cholate, 5 mM DTT, 5 mM MgCl2,1mMATP μ Protein Kinase Profiling (http://www.kinase-screen.mrc.ac.uk/) and 2 M PtdIns(4,5)P2 and incubated for 70 min at room μ as previously described [34]. Briefly, all assays were carried out temperature. Assays were stopped by addition of 5.5 l50mM ◦ μ robotically at room temperature (21 C) and were linear with EDTA and 25 l assay mixture was transferred to Lumitrac 200 μ respect to time and enzyme concentration under the conditions plate. Detection mix 1 was added to give a final volume of 50 l used. Assays were performed for 30 min using Multidrop Micro and the mixture was incubated for 20 min at room temperature reagent dispensers (Thermo Electron Corporation, Waltham, before reading. β β MA, U.S.A.) in a 96-well format. The abbreviations for each Class 2 PI3KC2 was assayed as follows: PI3KC2 (diluted kinase are defined in legend to Table 1. The concentration of in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, magnesium acetate in the assays was 10 mM and [γ -33P]ATP 1 mM DTT, 0.5 mM EGTA and 0.02% CHAPS) was assayed μ (∼800 c.p.m./pmol) was used at 5 μMforCK2α, DYRK3, against phosphoinositide substrate in a final volume of 50 l EF2K, ERK1, ERK8, GSK3β, HIPK2, IGF1R, IRR, MARK3, containing 19 mM Tris/HCl, pH 7.5, 143 mM NaCl, 0.96 mM MKK1, p38γ MAPK (mitogen-activated protein kinase), p38δ EDTA, 0.96 mM DTT, 0.48 mM EGTA, 0.02% CHAPS, μ MAPK, PAK4, PIM2, Akt1, , PKCζ and PRK2; 20 μMfor 20 M phosphatidylinositol, 0.2 mM ATP and 2 mM MgCl2 and CaMKKβ, CDK2/cyclin A, CHK1, CHK2, CK1δ,CSK,EPH-B3, incubated for 30 min at room temperature. Assays were stopped μ μ FGF-R1, IR, JNK1α1, JNK2α2, MAPKAP-K2, MSK1, MST2, by addition of 5.5 l50mMEDTAand2% CHAPS, and 25 l MST4, p38β MAPK, PKA, PAK5, PAK6, PDK1, PIM1, PIM3, was transferred to Lumitrac 200 plate. Detection mix 2 (18.6 mM PKCα, ROCKII, PRAK, S6K1, SGK1, SYK, VEGFR and YES1; Tris/HCl, pH 7.5, 140 mM NaCl, 0.9 mM DTT, 0.02% CHAPS, μ μ μ or 50 μM for AMPK, BRSK2, BTK, CaMK1, DYRK1a, DYRK2, 1.4 gSGKPX,0.06 MPtdIns3P–biotin, 1.6 g streptavidin– μ EPH-A2, ERK2, IKKε, LCK, MELK, NEK2A, NEK6, p38α, allophycocyanin and 0.64 g/ml Eu chelate-labelled antibody) PhKγ 1, Akt2, PKD1, RSK1, RSK2, SRPK1 and TBK1, in order was added to give a final volume of 50 ml and incubated for to be at or below the K for ATP for each enzyme [34]. 30 min at room temperature before reading. m Class 3 VPS34 was assayed as follows: VPS34 (diluted in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, Lipid kinase panel 1 mM DTT, 0.5 mM EGTA and 0.02% CHAPS) was assayed SPHK1 (sphingosine kinase 1) was assayed as follows: SPHK1 against phosphoinositide substrate in a final volume of 50 μl [diluted in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM containing 19 mM Tris/HCl, pH 7.5, 143 mM NaCl, 0.96 mM MgCl2, 1 mM EGTA and 1 mM DTT (dithiothreitol)] was assayed EDTA, 0.96 mM DTT, 0.48 mM EGTA, 0.02% CHAPS, μ against sphingosine in a final volume of 50 l containing 50 mM 20 μM phosphatidylinositol, 0.2 mM ATP and 2 mM MnCl2 and Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2,1mMEGTA, incubated for 60 min at room temperature. Assays were stopped 10 μM sphingosine, 10 μM ATP and 1 mM DTT and incubated by addition of 5.5 μl50mMEDTAand2% CHAPS and 25 μl for 30 min at room temperature. was transferred to a Lumitrac 200 plate. Detection mix 2 was SPHK2 was assayed as follows: SPHK2 (diluted in 50 mM added to give a final volume of 50 ml and incubated for 30 min at Tris/HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2,1mMEGTAand room temperature before reading. 1 mM DTT) was assayed against sphingosine in a final volume of 50 μl containing 50 mM Tris/HCl, pH 7.5, 200 mM KCl, mTOR complexes kinase assays 5mMMgCl2,1mMEGTA,10μM sphingosine, 1 μMATPand 1 mM DTT and incubated for 30 min at room temperature. HEK-293 cells were freshly lysed in Hepes lysis buffer. Lysate Choline kinase was assayed as follows: choline kinase (diluted (1–4 mg) was pre-cleared by incubating with 5–20 μl of Protein in 25 mM glycine/NaOH, pH 8.5, 67 mM KCl and 5 mM MgCl2) G–Sepharose conjugated to pre-immune IgG. The lysate extracts

c The Authors Journal compilation c 2009 Biochemical Society 32 J. M. Garc´ıa-Mart´ınez and others

Table 1 Effect of Ku-0063794 on 76 protein kinases Results are presented as percentage of kinase activity compared with control incubations, in which Ku-0063794 was omitted. Protein kinases were assayed as described in the Materials and methods + section. The results are an average of a triplicate determination − S.D. Asterisks indicate inhibition of >2-fold. Abbreviations not defined in main text: BRSK, brain-specific kinase; BTK, Bruton’s tyrosine kinase; CaMK1, -dependent kinase; CaMKK, CaMK kinase; CDK, cyclin-dependent kinase; CHK, checkpoint kinase; CK1, ; CSK, C-terminal Src kinase; DYRK, dual-specificity tyrosine-phosphorylated and regulated kinase; EF2K, elongation-factor-2 kinase; EPH, ephrin; FGF-R, fibroblast growth factor receptor; GSK3, synthase kinase 3; HIPK, homeodomain-interacting protein kinase; IGF1R, IGF1 receptor; IKK, inhibitory κB kinase; IR, insulin receptor; IRR, insulin-related receptor; JNK, c-Jun N-terminal kinase; Lck, lymphocyte cell-specific protein tyrosine kinase; MAPKAP-K, MAPK-activated protein kinase; MARK, microtubule-affinity-regulating kinase; MELK, maternal embryonic leucine-zipper kinase; MKK1, MAPK kinase-1; MLCK, smooth muscle myosin light-chain kinase; MNK, MAPK-integrating protein kinase; MSK, mitogen- and stress-activated protein kinase; MST, mammalian homologue Ste20-like kinase; NEK, NIMA (never in mitosis in Aspergillus nidulans)-related kinase; PAK, p21-activated protein kinase; PHK, ; PIM, provirus integration site for Moloney murine leukaemia virus; PKA, cAMP-dependent protein kinase; PKD, protein kinase D; PLK, polo-like kinase; PRAK, p38-regulated activated kinase; PRK, protein kinase C-related kinase; ROCK, Rho-dependent protein kinase; SRPK, -arginine protein kinase; SYK, spleen tyrosine kinase; TBK1, TANK-binding kinase 1; VEGFR, vascular endothelial growth factor receptor; YES1, Yamaguchi sarcoma viral oncogene homologue 1. n.d., not determined.

Percentage of activity remaining Percentage of activity remaining Kinase Ku-0063794 (1 μM) Ku-0063794 (10 μM) Kinase Ku-0063794 (1 μM) Ku-0063794 (10 μM)

+ + + + Akt1 98 − 890− 1 MLCK 82 − 4 104 − 3 + + + + Akt2 97 − 8 107 − 9 MNK1 111 − 075− 12 + + + + AMPK 97 − 1 106 − 3MNK2α 87 − 592− 1 + + + Aurora B n.d. 86 − 6 MSK1 101 − 6 117 − 7 + + + + BRSK2 90 − 172− 7MST281− 10 102 − 3 + + + + BTK 85 − 781− 21 MST4 87 − 586− 8 + + + + CAMK1 96 − 1 101 − 6 NEK2A 92 − 10 86 − 2 + + + + CAMKKβ 108 − 594− 0 NEK6 74 − 483− 10 + + + + CDK2/Cyclin A 89 − 590− 6 p38α MAPK 101 − 696− 5 + + + + CHK1 87 − 886− 7 p38β MAPK 106 − 783− 5 + + + + CHK2 97 − 296− 9 p38γ MAPK 89 − 891− 1 + + + + CK1δ 112 − 15 109 − 12 p38δ MAPK 72 − 187− 4 + + + + CK2α 104 − 085− 12 PAK4 110 − 175− 4 + + + + CSK 90 − 199− 3 PAK5 101 − 181− 3 + + + + DYRK1a 94 − 10 80 − 7 PAK6 75 − 3 100 − 24 + + + + DYRK2 92 − 196− 12 PDK1 89 − 591− 2 + + + + DYRK3 88 − 984− 1PhKγ 189− 2 116 − 3 + + + + EFK2 88 − 7 108 − 0PIM189− 498− 15 + + + + EPH-A2 108 − 896− 2PIM286− 11 78 − 0 + + + + EPH-B3 100 − 878− 4 PIM3 114 − 12 75 − 7 + + + + ERK1 102 − 087− 1 PKA 115 − 785− 12 + + + + ERK2 102 − 892− 9 PKCα 106 − 13 86 − 18 + + + + ERK8 88 − 192− 5 PKCζ 87 − 984− 0 + + + + FGF-R1 102 − 887− 10 PKD1 80 − 4 101 − 3 + + + + GSK3β 95 − 11 91 − 5PLK176− 294− 2 + + + + HIPK2 89 − 589− 1 PRAK 67 − 487− 8 + + + + IGF1R 117 − 089− 1 PRK2 95 − 5 105 − 12 + + + + IKKβ 104 − 389− 3 ROCKII 75 − 486− 5 + + + + IKKε 99 − 1 100 − 5 RSK1 100 − 486− 2 + + + + IR 81 − 167− 5 RSK2 103 − 7 104 − 1 + + + + IRR 105 − 771− 4 S6K1 104 − 11 98 − 4 + + + + JNK1α180− 593− 4SGK176− 664− 6 + + + JNK2α2 101 − 4 103 − 13 Src n.d. 86 − 5 + + + + LCK 69 − 5 104 − 9 SRPK1 101 − 194− 2 + + + + MAPKAP-K2 98 − 381− 10 SYK 88 − 383− 5 + + + + MARK3 93 − 693− 15 TBK1 97 − 889− 4 + + + + MELK 91 − 672− 4 VEGFR 92 − 892− 8 + + + + MKK1 96 − 443− 7* YES1 94 − 10 90 − 1

were then incubated with 5–20 μl of Protein G–Sepharose ATP and 10 mM MgCl2 in the presence or absence of Ku- conjugated to 5–20 μg of either anti-Rictor or anti-Raptor 0063794 and GST–Akt1 (0.5 μg) or GST–S6K1 (0.5 μg). antibody, or pre-immune IgG. All antibodies were covalently Reaction were carried out for 30 min at 30 ◦C on a vibrating conjugated to Protein G–Sepharose. Immunoprecipitations were platform and stopped by addition of SDS sample buffer. carried out for 1 h at 4 ◦C on a vibrating platform. The Reaction mixtures were then filtered through a 0.22-μm-pore- immunoprecipitates were washed four times with Hepes lysis size Spin-X filter and samples were subjected to electrophoresis buffer, followed by two washes with Hepes kinase buffer. For and immunoblot analysis. Raptor immunoprecipitates used for phosphorylating S6K1, for the initial two wash steps the buffer included 0.5 M NaCl Kinase assays to ensure optimal kinase activity [7]. GST–Akt1 was isolated HEK-293 were lysed in Tris lysis buffer. In order to perform from serum-deprived HEK-293 cells incubated with PI-103 Akt and S6K assays, 500 μg of lysate was incubated with (1 μM for 1 h) [24]. GST–S6K1 was purified from serum- 5 μg of the corresponding antibody conjugated to Protein G– deprived HEK-293 cells incubated with rapamycin (0.1 μMfor Sepharose. To perform SGK1 activity assays, 50 μg of transfected 1 h) [25]. mTOR reactions were initiated by adding 0.1 mM lysate was incubated with 5 μg of glutathione–Sepharose. All

c The Authors Journal compilation c 2009 Biochemical Society Novel small molecule mTOR inhibitor 33

Figure 1 Ku-0063794 inhibits both mTORC1 and mTORC2 complexes in vitro

(A) Structure of Ku-0063794. (B and C) HEK-293 cell extracts were subjected to immunoprecipitation (IP) with pre-immune (IgG), anti-Raptor (B) or anti-Rictor antibody (C). The immunoprecipitates were incubated with dephosphorylated GST–S6K1 (B) or GST–Akt1 (C) in the presence of the indicated concentrations of Ku-0063794 or vehicle (DMSO). Kinases assays were performed for 30 min in the presence of MgATP and then subjected to immunoblot analysis with the indicated antibodies. Similar results were obtained in three independent experiments. T389-P, phosphorylated Thr389; S473-P, phosphorylated Ser473. the incubations were performed for 1 h at 4 ◦C on a vibrating 24 h with freshly dissolved inhibitors. Every 24 h of treatment, platform. Kinase activity was assayed exactly as described cells were washed once with PBS, and fixed in 4% (v/v) previously [35] using the Crosstide peptide (GRPRTSSFAEG) paraformaldehyde in PBS for 15 min. After washing once with at 30 μM. Incorporation of [32P] into the peptide water, the cells were stained with 0.1% Crystal Violet in 10% substrate was determined by applying the reaction mixture to ethanol for 20 min and washed three times with water. Crystal P81 phosphocellulose paper and liquid-scintillation counting of Violet was extracted from cells with 0.5 ml of 10% (v/v) ethanoic radioactivity after washing the papers in phosphoric acid. One unit (acetic) acid for 20 min. The eluate was then diluted 1:10 in water, of activity was defined as that which catalysed the incorporation and absorbance at 590 nm was quantified. of 1 nmol of [32P]phosphate into the substrate. Flow cytometric analysis of cell cycle distribution GST-pull down of transfected SGK1 for immunoblotting analysis Adherent MEFs were harvested by trypsinization, washed once At 36 h post-transfection, HEK-293 cells were lysed in Tris in PBS, and re-suspended in ice-cold aq. 70% (v/v) ethanol. lysis buffer. Lysate (0.5 mg) was affinity-purified on glutathione– Cells were washed twice in PBS plus 1% (w/v) BSA and stained Sepharose. Incubations were carried out for 1 h at 4 ◦Cona for 20 min in PBS plus 0.1% (v/v) Triton X-100 containing vibrating platform. The resultant precipitates were then washed 50 g/ml propidium iodide and 50 g/ml RNase A. The DNA content twice with Tris lysis buffer containing 0.5 M NaCl, followed of cells was determined using a FACSCalibur flow cytometer by two washes with Buffer A. The immunoprecipitates were (BD Biosciences) and CellQuest software. Red fluorescence resuspended in 20 μl of sample buffer, filtered through a 0.22-μm- (585 −+ 42 nm) was acquired on a linear scale, and pulse width pore-size Spin-X filter, and samples were subjected to analysis was used to exclude doublets. Cell-cycle distribution electrophoresis and immunoblot analysis as described below. was determined using FlowJo software (Tree Star).

Immunoblotting RESULTS Total cell lysate (5–20 μg) or immunoprecipitated samples were Ku-0063794 is a specific mTORC1 and mTORC2 inhibitor heated at 95 ◦C for 5 min in sample buffer, and subjected to PAGE and electrotransfer to nitrocellulose membrane. Membranes were The structure of Ku-0063794 is shown in Figure 1(A). blocked for 1 h in TBS-Tween buffer containing 10% (w/v) non- It inhibited the activity of endogenous immunoprecipitated fat dried skimmed milk powder. The membranes were probed mTORC1, assayed employing S6K1 as substrate (Figure 1B) and mTORC2, assayed using Akt as substrate (Figure 1C) with with the indicated antibodies in TBS-Tween containing 5% (w/v) ∼ skimmed milk or 5% (w/v) BSA for 16 h at 4 ◦C. Detection was an IC50 of 10 nM. To evaluate the specificity of Ku-0063794, performed using horseradish peroxidase-conjugated secondary we studied the effect of Ku-0063794 at ATP concentrations, antibodies and the enhanced chemiluminescence reagent. which approximate to the Km constant for ATP, towards a panel of 76 protein kinases. At 1 μM Ku-0063794, which completely suppressed mTORC1 and mTORC2 activity (Figures 1B and 1C), Cell growth none of the kinases on the specificity panel were significantly MEFs were seeded in 24-well plates (20000 cells per 1.91 cm2 inhibited (Table 1). Even at 10 μM, the only enzyme on the well) and grown overnight in the presence of 10% foetal bovine panel that was inhibited more than 2-fold was MAPK kinase-1, serum. Cells were then treated in the presence or absence or which was decreased ∼55% (Table 1). Ku-0063794, even at rapamycin or Ku-0063794 and the medium was changed every concentrations of 10 μM, did not significantly inhibit seven lipid

c The Authors Journal compilation c 2009 Biochemical Society 34 J. M. Garc´ıa-Mart´ınez and others

Table 2 Effect of Ku-0063794 on seven lipid kinases Ku-0063794 inhibits mTORC2 activity in HEK-293 cells Results are presented as percentage of lipid kinase activity compared with control incubations To investigate whether Ku-0063794 inhibited mTORC2, we in which Ku-0063794 was omitted. Lipid kinases were assayed as described in the Materials studied how it affected Akt activity and hydrophobic motif (Ser473) and methods section, in the absence or presence of the indicated concentration of Ku-0063794. The results are an average of a triplicate determinations + S.D. phosphorylation in HEK-293 cells. In the presence of serum − (Figure 3A) or following IGF1 stimulation (Figure 3B), Ku- Percentage of activity remaining 0063794 caused a dose-dependent suppression of Akt activity, accompanied by inhibition of Ser473 phosphorylation. As observed μ μ Kinase Ku-0063794 (1 M) Ku-0063794 (10 M) with S6K1, the activity and phosphorylation of Akt was inhibited by lower concentrations of Ku-0063794 when cells PI3Ks were cultured in serum compared with stimulation with IGF1 Class I + + (compare Figure 3A with Figure 3B). IGF1 induces a ∼6- PI3Kα 97 − 295− 3 + + PI3Kβ 95 − 984− 22 fold greater activation of Akt than serum (compare Figure 3A Class II with Figure 3B), which is likely to account for the higher + + PI3KC2β 126 − 13 82 − 12 concentrations of Ku-0063794 required to inhibit Akt and S6K Class III + + in IGF1-stimulated cells compared with serum. The effects of VPS34 101 − 493− 1 Ku-0063794 on Akt activity and phosphorylation were observed Other lipid kinases + + within 10 min and were sustained for up to 48 h (Figures 3C Choline kinase 102 − 1 102 − 1 + + and 3D). Phosphorylation of the Akt substrates PRAS40 at SPHK1 108 − 399− 7 + + Thr246 [39], GSK3α/GSK3β at Ser21/Ser9 [40] and Foxo-1/3a SPHK2 119 − 2 116 − 3 at Thr24/Thr32 [41] was also inhibited to a similar extent by Ku- 0063794 as Akt activity and phosphorylation in cells cultured kinases tested (Class I PI3Kα and PI3Kβ, Class II PI3K-B, Class in serum (Figure 3A). In cells stimulated with IGF1, which induced a greater activation of Akt than observed with serum, III VPS34 as well as SPHK-1, SPHK-2 and choline kinase) μ (Table 2). even concentrations of 1 M Ku-0063794 did not inhibit Akt phosphorylation and activation completely (Figure 3B). At 1 μM Ku-0063794 in IGF-1 stimulated cells, Akt1 activity was reduced Ku-0063794 inhibits mTORC1 activity in HEK-293 cells to ∼0.5 units/mg, which is ∼50% of the Akt activity observed with cells cultured in serum (Figure 3A). This may explain why We next investigated the effect of adding increasing amounts phosphorylation of GSK3 and Foxo are not completely suppressed of Ku-0063794 on the activity and phosphorylation of S6K1 in with 1 μM Ku-00637954 in IGF1-stimulated compared with HEK-293 cells cultured in the presence of serum (Figure 2A). serum-treated cells (compare Figure 3A with Figure 3B). Under these conditions, with a concentration of Ku-0063794 as We also studied the effect that Ku-0063794 had on the turn- low as 30 nM, Ku-0063794 almost ablated S6K1 activity and motif Thr450 residue of Akt1, whose phosphorylation is regulated phosphorylation of the hydrophobic motif (Thr389). Consistent by mTORC2 [26,27]. Even after 48 h treatment with 1 μMKu- with phosphorylation of the T-loop of S6K1 being dependent upon 0063794, phosphorylation of Akt at Thr450 was only moderately prior phosphorylation of the hydrophobic motif, Ku-0063794 reduced (Figure 3D). This is consistent with phosphorylation of inhibited phosphorylation of the T-loop residue (Thr229). Ku- Thr450 being introduced in Akt shortly after its synthesis and 0063794 also inhibited phosphorylation of the ribosomal S6 turning over very slowly [26,27]. protein, an S6K substrate (Figure 2A). The ability of Ku-0063794 to suppress S6K1 activity and phosphorylation was rapid, with maximal inhibition seen within 10 min and sustained for at least 308 4 h, the longest time examined (Figure 2B). Ku-0063794 also Ku-0063794 inhibits phosphorylation of Akt at Thr in HEK-293 suppressed S6K1 activity and phosphorylation induced by IGF1 cells stimulation of serum-starved HEK-293 cells, although higher We observed that, in addition to inhibiting Akt Ser473 phos- concentrations were required to fully inhibit S6K1, compared with phorylation, Ku-0063794 suppressed phosphorylation of Akt cells cultured in serum (compare Figure 2A with Figure 2C). For at Thr308 (Figures 3A–3D). This was unexpected, as some example, in the presence of serum, 30 nM Ku-0063794 suppressed previous work suggested that the phosphorylation of Thr308 by S6K1 activity by ∼90%, whereas following IGF1 stimulation, PDK1 is not directly influenced by mTORC2. Ku-0063794 30 nM Ku-0063794 suppressed S6K1 activity by ∼50%.Inthe was not suppressing Thr308 phosphorylation by inhibiting PI3K presence of IGF1, a concentration of 300 nM Ku-0063794 was or PtdIns(3,4,5)P3 production, as IGF1 induced a similar necessary to reduce S6K1 activity ∼90%. mTORC1, and hence enhancement of PtdIns(3,4,5)P3 levels in the absence or pre- S6K1, is also activated by nutrients such as amino acids. We sence of 1 μM Ku-0063794, under conditions which the observed that Ku-0063794 induced a dose-dependent inhibition PI3K inhibitor PI-103 ablated IGF1-induced PtdIns(3,4,5)P3 of the phosphorylation of S6K1 and S6 protein induced following production (Figure 4A). To further evaluate the effect that the replenishment of amino acids to HEK-293 cells incubated Ku-0063794 had on Thr308 phosphorylation, we overexpressed in medium lacking these nutrients (Figure 2D). Concentrations in HEK-293 cells a mutant form of GST–Akt1 in which of 100–300 nM Ku-0063794 were required to fully suppress Ser473 was changed to aspartate in an attempt to mimic the amino-acid-induced phosphorylation of S6K1 and S6 protein Akt-phosphorylated form of mTORC2 and tested how Ku- (Figure 2D). 0063794 affected phosphorylation at Thr308. We found that Recent work has shown that mTORC1 is phosphorylated at IGF1-induced phosphorylation of GST–Akt1(S473D) at Thr308 Ser2448 [36], a reaction probably mediated by S6K1 [37,38]. was not suppressed by Ku-0063794 to the same extent as In contrast, mTORC2 autophosphorylates at Ser2481 [36]. We endogenous Akt in these cells, suggesting that Ku-0063794 is observed that Ku-0063795 suppressed phosphorylation of both not directly inhibiting PDK1-mediated phosphorylation of Thr308 Ser2448 and Ser2481 in a dose-dependent and time-dependent (Figure 4B). In parallel studies, Ku-0063794 also suppressed manner, similar to S6K1 (lower panels of Figures 2A–2C). Thr308 phosphorylation of wild-type GST–Akt1 to a greater extent

c The Authors Journal compilation c 2009 Biochemical Society Novel small molecule mTOR inhibitor 35

Figure 2 Ku-0063794 inhibits mTORC1 activity in vivo

(A and B) HEK-293 cells cultured in the presence of 10% foetal bovine serum, were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 (A)orwith1μM Ku-0063794 for the indicated time (B). Cells were lysed and S6K1 immunoprecipitated and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific + activity − S.E.M. from three different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. (C)Asin(A), except that cells were deprived of serum for 16 h prior to stimulation with 50 ng/ml of IGF-1 for 20 min. (D)Asin(C), except that, after 16 h of serum deprivation, cells were incubated for 1 h in amino-acid-free EBSS (Earle’s Balanced Salt Solution) medium containing 10% dialysed serum. Cells were then incubated in the absence or presence or the indicated amount of Ku-0063794 for 30 min prior to re-addition of physiological levels of amino acids for an additional 30 min. Immunoblots are representative of three different experiments. than GST–Akt1(S473D) (Figure 4B). The dual PI3K and mTOR SGK1 activity and hydrophobic motif phosphorylation should inhibitor PI-103 inhibited phosphorylation of Thr308 on both GST– therefore be inhibited by Ku-0063794, but not rapamycin. Akt1(S473D) and wild-type Akt1 (Figure 4B). We also examined As it is not possible to assay phosphorylation and activity the effect that Ku-0063794 had on the phosphorylation of Akt at of endogenous SGK1 [21], we investigated how Ku-0063794 Thr308 in MEFs lacking the critical mTORC2 subunits Rictor [29] affected activity and phosphorylation of overexpressed full-length (Figure 4C), mLST8 [17] (Figure 4D) and Sin1 [16] (Figure 4E). SGK1 in HEK-293 cells cultured in the presence of serum Consistent with previous work, IGF1 induced phosphorylation of (Figure 5A). We observed that Ku-0063794 inhibited SGK1 Thr308, but not Ser473, in mTORC2-deficient cells (Figures 4C– activity and Ser422 phosphorylation in a dose-dependent manner, 4E). However, Ku-0063794 did not inhibit phosphorylation of to the same extent as Akt and S6K1 phosphorylation (Figure 5A). Thr308 in any of the mTORC2-deficient cells under conditions We also examined the phosphorylation of NDRG1, a well- where it suppressed Thr308 phosphorylation in control wild-type characterized substrate for SGK1, which is not phosphorylated cells (Figures 4C–4E). Treatment of mTORC2-deficient cells with in SGK1 knockout mouse tissues [44]. Employing an antibody PI-103 abolished phosphorylation of Thr308. recognizing the SGK1-phosphorylated form of NDRG1, we found that Ku-0063794 inhibited NDRG1 phosphorylation to the same extent as it suppressed SGK1 activity. Phosphorylation of Ku-0063794 inhibits SGK1 but not RSK endogenous NDRG1 was also inhibited by Ku-0063794 in HeLa We have shown that SGK1 activity and phosphorylation of (Figure 5B) cells as well as wild-type MEFs (Figure 5C). The its hydrophobic motif (Ser422) is regulated by mTORC2 but dual PI3K/mTOR inhibitor PI-103, but not rapamycin or the Akt not mTORC1 [21]. This finding has recently been supported inhibitor Akti-1/2 [45], reduced phosphorylation of NDRG1 in by elegant genetic studies in Caenorhabditis elegans [42,43]. both HeLa cells and wild-type MEFs.

c The Authors Journal compilation c 2009 Biochemical Society 36 J. M. Garc´ıa-Mart´ınez and others

Figure 3 Ku-0063794 ablates mTORC2 in vivo

HEK-293 cells were cultured in serum, and stimulated with IGF-1 in the absence or presence of Ku-0063794 as described in the legend to Figure 2. Akt1 was immunoprecipitated and catalytic activity + assessed employing the Crosstide substrate. Each bar represents the mean specific activity − S.E.M. from three different samples, with each sample assayed in duplicate. Cell lysates were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. In (D), medium containing freshly dissolved Ku-0063794 was replaced every 12 h. P, phosphorylated.

To ensure Ku-0063794 was not inhibiting the phosphorylation Ku-0063794 suppresses cell growth and induces G1-cell cycle and activity of all AGC kinases, we studied the effect that arrest Ku-0063794 had on the activation of the RSK, which is We compared the effect that Ku-0063794 and rapamycin had on activated by ERK1/ERK2 pathway and not regulated by mTOR. growth of wild-type and mLST8 deficient MEFs. We selected HEK-293 cells were stimulated with phorbol ester, which these cells, as despite lacking mTORC2, they proliferate at markedly enhanced ERK as well as RSK phosphorylation and similar rates in serum as wild-type control fibroblasts. Moreover, activity (Figure 5D). Ku-0063794 did not inhibit phorbol ester- mLST8-fibroblast cell lines proliferate more rapidly than Rictor- induced ERK or RSK phosphorylation and RSK activation under or Sin1-deficient MEFs (results not shown). We observed that conditions in which the MAPK kinase inhibitor PD 0325901- Ku-0063794 suppressed growth of both wild-type (Figure 7A) CL [46] ablated ERK phosphorylation and RSK activation and mLST8-deficient (Figure 7C) MEFs more markedly than (Figure 5D). rapamycin. Flow-cytometric analysis indicated that Ku-0063794 increased the proportion of wild-type (Figure 7B) and mLST8

Ku-0063794 induces complete dephosphorylation of 4E-BP1 knockout MEFs (Figure 7D) in the G1 cell cycle state approx. 2- fold, compared with rapamycin, which increased the proportion Consistent with mTOR regulating 4E-BP1 phosphorylation, we of cells in G by 1.5-fold. found that treatment of wild-type MEFs (Figure 6A) or HEK-293 1 cells (Figure 6B) with Ku-0063794 induced a marked increase in the electrophoretic mobility of 4E-BP1, which was accompanied DISCUSSION by complete dephosphorylation of Thr37,Thr46 and Ser65.We observed that rapamycin reduced phosphorylation of 4E-BP1 and Our results from the present study establish that Ku-0063794 enhanced electrophoretic mobility to a significantly lower extent is a potent and highly specific inhibitor of mTOR as it does than Ku-0063794, even in mTORC2-deficient Sin1 knockout not significantly inhibit 76 other protein kinases tested as well MEFs (Figures 6A and 6B). as seven lipid kinases, including class 1a PI3Kα and PI3Kβ.

c The Authors Journal compilation c 2009 Biochemical Society Novel small molecule mTOR inhibitor 37

Figure 4 Further investigation of the effect of Ku-0063794 on Akt Thr308 phosphorylation

(A) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103, then stimulated with 50 ng/ml of IGF-1 for + 10 min. Cell were lysed with 0.5 M trichloroacetic acid and amounts of PtdIns(3,4,5)P3 were determined. Each bar represents the means − S.E.M. from three independent samples. (B) HEK-293 cells were transfected with a DNA construct encoding wild-type or mutant GST–Akt1. At 24 h post-transfection, cells were deprived of serum for 16 h and stimulated with IGF-1 for 20 min, in the presence or absence of inhibitors as described in (A). Cell lysates were analysed by immunoblotting with the indicated antibodies and immunoblots shown are representative of three separate experiments. (C–E) The indicated wild-type (wt) or knockout (ko) MEFs were deprived and stimulated with IGF-1 for 20 min, in the presence or absence of inhibitors as described in (A). Cell lysates were analysed by immunoblotting with the indicated antibodies. Similar results were obtained in three independent experiments. P, phosphorylated. mTOR belongs to the PI3K-related kinases (PIKK) subgroup, as PI3K inhibitors have been described that do not inhibit mTOR, it possesses significant similarity to PI3Ks [47]. Many widely such as GDC-0941 [51]. Our results from the present study used PI3K inhibitors, including wortmannin [48], LY294002 demonstrate that Ku-0063794 does not inhibit PI3K in vivo,as [48], PI-103 [49] and NVP-BEZ235 [50], inhibit mTOR with a Ku-0063794 does not suppress increases in cellular levels of potency similar to that towards Class 1 PI3Ks. Recently, specific PtdIns(3,4,5)P3 following IGF-1 stimulation of HEK-293 cells

c The Authors Journal compilation c 2009 Biochemical Society 38 J. M. Garc´ıa-Mart´ınez and others

Figure 5 Ku-0063794 suppresses hydrophobic motif phosphorylation and activation of SGK1 but not RSK

(A) HEK-293 cells were transfected with a DNA construct encoding GST–SGK1 (full-length enzyme). Cells were cultured in the presence of 10% foetal bovine serum in order to maintain PI3K pathway activity. At 36 h post-transfection, cells were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103. Cells were lysed, SGK1 was affinity-purified + on glutathione–Sepharose and catalytic activity was assessed employing the Crosstide substrate. Each bar represents the mean specific activity − S.E.M. from three different samples, with each sample assayed in duplicate. Affinity purified SGK1 was also subjected to immunoblotting with an anti-GST antibody (SGK1-Total) as well as an anti-Ser422 phosphospecific antibody (S422-P). Cell lysates were also analysed by immunoblotting with the indicated non-SGK antibodies. (B and C) HeLa cells or the indicated wild-type (wt) or knockout (ko) MEFs were cultured in the presence of 10% serum, then treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. (D) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of 1 μM Ku-0063794 or 0.2 μM PD 0325901 then stimulated with 400 ng/ml of PMA for 15 min. RSK was immunoprecipitated with an antibody recognizing all isoforms and catalytic activity assessed employing the + Crosstide substrate. Each bar represents the mean specific activity − S.E.M. from two different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. P, phosphorylated.

(Figure 4A). Moreover, Ku-0063794 does not inhibit Akt Thr308 inhibitors will be useful in confirming specificity of physiological phosphorylation in three different mTORC2-deficient MEF cell responses observed when using these reagents. Our findings using lines (Figures 4C–4E) under conditions which PI-103 inhibited Ku-0063794 are similar to those reported for the PP242 [52] and 308 PtdIns(3,4,5)P3 production and Thr phosphorylation. Torin [53], as all three inhibitors reduced hydrophobic motif as Two other mTOR specific inhibitors that do not inhibit PI3K well as T-loop phosphorylation of endogenous S6K as well as Akt. have recently been described, namely PP242 [52] and Torin [53]. We also demonstrated Ku-0063794, but not rapamycin or Akti- Although the chemical structure of Torin has thus far not been 1/2, inhibited phosphorylation and activation of SGK1 as well disclosed, the chemical structures of PP242 and Ku-0063794 as its physiological substrate NDGR1. This is consistent with our are quite distinct. We have synthesized PP242 and compared its findings that SGK1 is regulated by mTORC2 and not the mTORC1 specificity with Ku-0063794 and observed that it is less selective. inhibitor rapamycin (reviewed in [54]). We would expect Torin At 1 μM PP242, 22 of the 76 kinases tested were inhibited by and PP242 to also inhibit SGK1 activation and phosphorylation. >50%, whereas at 10 μM PP242, 46 out of the 76 kinases The finding that Ku-0063794 as well as Torin [53] and PP242 were inhibited (results available upon request from D.R.A.). [52] inhibited Akt Thr308 phosphorylation is arguably unexpected, In contrast, 10 μM Ku-0063794 only suppressed 1 out of 76 as previous work suggested that Thr308 and Ser473 phosphorylation kinases over 2-fold (Table 1). Torin was reported to be highly can occur independently from each other. The initial evidence selective [53]. The availability of chemically unrelated mTOR for this was that an Akt-T308A mutant overexpressed in

c The Authors Journal compilation c 2009 Biochemical Society Novel small molecule mTOR inhibitor 39

PDK1 PIF-pocket mutant knockin cells, Akt activation may be so essential for survival that signalling networks adapt so that Akt can be phosphorylated by PDK1 at Thr308 independently of Ser473 phosphorylation. Evidence to support the idea that PDK1 might interact with Ser473 after its phosphorylation by mTORC2 might stem from recent observations made in PDK1- PH domain knockin mice that express a form of PDK1 that

can not interact with PtdIns(3,4,5)P3. In tissues or cells derived from these animals, Akt is still phosphorylated at Thr308,albeit to ∼3-fold lower level than in wild-type tissues or cells [62]. This observation was unexpected, as previous evidence indicated that PDK1 was needed to interact with phosphoinositides at the plasma membrane in order to phosphorylate Akt at Thr308 [19]. However, if phosphorylation at Ser473 by mTORC2 does stimulate interaction of PDK1 with Akt, this may bypass the need for PDK1

to interact with PtdIns(3,4,5)P3 and enhance phosphorylation of Thr308 without the need of PDK1 binding to phosphoinositides. In future work, it would important to investigate the effect on Akt Thr308 phosphorylation of allosteric PIF-pocket PDK1 inhibitors being elaborated [63,64] or mTORC2-specific inhibitors (if such compounds could be developed). Another possibility to account for the effect of mTOR inhibitors on Akt Thr308 phosphorylation is if the lack of Akt Ser473 phosphorylation leads to destabilization of Akt, promoting de- phosphorylation of Thr308 by (s). Indeed, this idea is supported by structural analysis of Akt2, where it has been demonstrated that hydrophobic motif phosphorylation induces a marked disorder-to-order transition of the kinase domain, resulting in stabilization of the alphaC helix with concomitant restructuring of the T-loop and reconfiguration of the structure of the catalytic domain [65]. Treatment of cells with Ku-0063794 will inhibit Akt Ser473 phosphorylation and may lead to less stable Figure 6 Ku-0063794 induces a marked dephosphorylation of 4E-BP1 Akt conformation, with Thr308 being more exposed and accessible (A and B) The indicated wild-type (wt) or knockout (ko) MEFs (A) or HEK-293 cells (B)were to becoming dephosphorylated by protein phosphatase(s). It cultured in the presence of 10% serum, and treated for 30 min in the absence or presence of the would be interesting to investigate whether in mTORC2-deficient indicated concentrations of inhibitors. Cells were lysed and extracts analysed by immunoblotting knockout cells, in which Thr308 phosphorylation is unaffected, with the indicated antibodies. Immunoblots are representative of three different experiments. P, the activity of the protein phosphatase(s) that act on Thr308 is phosphorylated. reduced, or if Akt is stabilized in some way that enables it to function without Ser473 phosphorylation. IGF1-stimulated HEK-293 cells was still phosphorylated at Ku-0063794 (Figure 6), as well as Torin [53] and PP242 Ser473 and vice versa [55]. Moreover, in cells lacking mTORC2 [52], induce a much greater dephosphorylation of 4E-BP1 than activity, Akt is still phosphorylated at Thr308 despite not being rapamycin, suggesting that these drugs will more effectively phosphorylated at Ser473 [16,17,29]. In PDK1-deficient cells suppress protein synthesis resulting from overactivation of the [56] and in mouse tissues lacking PDK1 [57,58], Akt is still PI3K/Akt pathways. Both mTORC1 and mTORC2 activity would phosphorylated at Ser473, but not at Thr308. One explanation for be inhibited in mTORC2-deficient cells treated with rapamycin. the effect that mTOR inhibitors such as Ku-0063794 have on However, we still observed significant phosphorylation of 4E- Akt Thr308 phosphorylation would be if Akt phosphorylation BP1 at Thr37/Thr46 and Ser65 in Sin-1 knockout MEFs treated of Ser473 generated a docking site for PDK1 to interact and with rapamycin, under conditions in which the hydrophobic motif phosphorylate Thr308, in the same way in which phosphorylation of both S6K and Akt are not phosphorylated (in agreement of the hydrophobic motifs of S6K/SGK by mTOR promotes with lack of mTORC1 and mTORC2 activity). In contrast, T-loop phosphorylation of these enzymes by PDK1. The evidence treatment of Sin-1 knockout MEFs with Ku-0063794 led to a against this stems from previous studies undertaken in knockin much greater dephosphorylation of 4E-BP1 than observed with cells [59,60] and in mouse tissues [61] that express a PIF- rapamycin. Similar results were also found with the Torin [53] pocket mutant form of PDK1 that is unable to interact with the and PP242 [52] inhibitors. Taken together, these results indicate hydrophobic motifs of S6K and SGK1. In these knockin studies, it that rapamycin may not inhibit the phosphorylation of 4E-BP1 was demonstrated that Akt was normally phosphorylated at Thr308, by mTORC1 and/or there may be a rapamycin-resistant form of under conditions which S6K and SGK are not phosphorylated at mTOR, distinct from mTORC2. The existence of such a complex their T-loop residue, suggesting that Akt Thr308 phosphorylation might also explain why Ku-0063794 inhibits proliferation of was not dependent on PDK1 interacting with Akt via its PIF- mTORC2-deficient mLST8 knockout cells more effectively than pocket. How can the effects on Thr308 phosphorylation in mTOR rapamycin (Figures 7C–7D). It would be interesting to see inhibitor and knockin/knockout studies be reconciled? One whether it might be possible to isolate a form of mTOR capable possibility is that in wild-type cells, phosphorylation of Akt at of phosphorylating 4E-BP1, which was inhibited by Ku-0063794, Ser473 does indeed promote PDK1 interaction and contributes but not by rapamycin. towards maximal phosphorylation of Thr308 by PDK1. However, There is great expectation that inhibitors of the PI3K-PDK1- in knockout cells permanently lacking mTORC2 activity or in Akt-mTOR signalling axis will have utility as anti-cancer drugs,

c The Authors Journal compilation c 2009 Biochemical Society 40 J. M. Garc´ıa-Mart´ınez and others

Figure 7 Ku-0063794 inhibits cell growth and induces a G1 cell cycle arrest

The indicated wild-type (wt) or knockout (ko) MEFs were passaged at low density (20000 cells per 1.91 cm2 dish) and cells were cultured for 1, 2 or 3 days in the absence (DMSO) or presence of 3 μM Ku-0063794 or 100 nM rapamycin. (A and C) Cell numbers were measured in quadruplicate using a Crystal Violet staining as described in the Materials and methods section. The results are + presented as means − S.E.M. of the percentage of cells relative to the DMSO control. Flow cytometric analysis was also undertaken as described in the Materials and methods section. (B and D) DNA was stained with propidium iodide, and cellular content was analysed. The percentage of cells in G1,S,orG2 phases were determined from triplicate dishes for each condition using CellQuest software. Two-way ANOVA and Bonferroni posttests were performed, *P < 0.001 compared with DMSO control. #P < 0.001 compared with rapamycin. as this signalling network is inappropriately activated in the of Dundee, Dundee, Scotland, U.K.] co-ordinated by Hilary McLauchlan and James Hastie majority of human cancers. Rapamycin and its analogues are for expression and purification of antibodies. being extensively deployed for the treatment of various cancers [66]. It will be interesting to evaluate whether an inhibitor such as Ku-0063794, which targets both mTOR complexes, is FUNDING a more effective anti-cancer agent than rapamycin. This might J.M.G.M. is funded by a grant from AstraZeneca. We also thank the Medical Research be expected, as mTOR inhibitors such as Ku-0063794, unlike Council for financial support. rapamycin derivatives, will inhibit Akt and SGK isoforms that are likely to play vital roles in driving the proliferation of many cancers. The finding that mTOR inhibitors such as Ku- REFERENCES 0063794 induce more marked dephosphorylation of 4E-BP1 than 1 Wullschleger, S., Loewith, R. and Hall, M. N. (2006) TOR signaling in growth and rapamycin also holds promise that these drugs will be more . Cell 124, 471–484 effective at suppressing protein synthesis required for growth 2 Sarbassov, D. D., Ali, S. M. and Sabatini, D. M. (2005) Growing roles for the mTOR and proliferation of cancer cells than rapamycin derivatives. Ku- pathway. Curr. Opin. Cell Biol. 17, 596–603 0063794 and other mTOR inhibitors will also be very useful 3 Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A., Crespo, J. L., Bonenfant, D., reagents to dissect and validate the molecular roles that mTOR Oppliger, W., Jenoe, P. and Hall, M. N. (2002) Two TOR complexes, only one of which is plays and hence extend our repertoire of knowledge of how this rapamycin sensitive, have distinct roles in cell growth control. Mol. 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A., Spooner, E., undertook FACS analysis and Alex Gray measured PtdIns(3,4,5)P 3 levels. Sabina C. Cosulich and Christine M. Chresta were involved in the development of Ku-0063794. Carr, S. A. and Sabatini, D. M. (2007) PRAS40 is an insulin-regulated inhibitor of the Juan M. Garc´ıa-Mart´ınez and Dario R. Alessi wrote the manuscript. mTORC1 protein kinase. Mol. Cell 25, 903–915 8 Nicklin, P., Bergman, P., Zhang, B., Triantafellow, E., Wang, H., Nyfeler, B., Yang, H., Hild, M., Kung, C., Wilson, C. et al. (2009) Bidirectional transport of amino acids ACKNOWLEDGEMENTS regulates mTOR and autophagy. Cell 136, 521–534 We thank Mark Magnuson, David Sabatini, and Bing Su for provision of reagents. We 9 Sancak, Y., Peterson, T. R., Shaul, Y. D., Lindquist, R. A., Thoreen, C. C., Bar-Peled, L. are also grateful to the Sequencing Service for DNA sequencing, the Post Genomics and Sabatini, D. M. (2008) The Rag GTPases bind raptor and mediate amino acid and Molecular Interactions Centre for Mass Spectrometry facilities (School of Life signaling to mTORC1. Science 320, 1496–1501 Sciences, University of Dundee, Dundee, Scotland, U.K.) and the protein production and 10 Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. and Guan, K. L. (2008) Regulation of antibody purification teams [Division of Signal Transduction Therapy (DSTT), University TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 10, 935–945

c The Authors Journal compilation c 2009 Biochemical Society Novel small molecule mTOR inhibitor 41

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Received 26 March 2009/23 April 2009; accepted 29 April 2009 Published as BJ Immediate Publication 29 April 2009, doi:10.1042/BJ20090489

c The Authors Journal compilation c 2009 Biochemical Society